Animal model for chronic obstructive pulmonary disease and cystic fibrosis

ABSTRACT

A nonhuman transgenic mammal is described whose genome comprises a promoter construct operably linked to a heterologous DNA encoding an epithelial sodium channel β subunit, wherein said promoter construct directs expression of the epithelial sodium channel β subunit in lung epithelial cells of said animal, and wherein said transgenic mammal has increased lung mucus retention as compared to the corresponding wild-type mammal. The animal is useful in screening compounds for activity in treating lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease.

STATEMENT OF FEDERAL SUPPORT

[0001] This invention was made with Government support under grantnumber HL 34322 from the National Institutes of Health. The Governmenthas certain rights to this invention.

FIELD OF THE INVENTION

[0002] The present invention concerns non-human transgenic animals thatare useful as models of lung diseases such as chronic obstructivepulmonary disease (COPD) and cystic fibrosis.

BACKGROUND OF THE INVENTION

[0003] Cystic fibrosis (CF) is among the most prevalent, lethal diseasesof genetic origin. Approximately 30,000 children and adults are affectedin the United States alone. In this disease, abnormal ion transportacross the respiratory epithelia leads to dehydrated, viscous andpoorly-cleared airway secretions that contribute to chronic infection ofthe airways and early death. Knowles, Clin. Chest. Med. 11, 75 (1986).Chronic obstructive pulmonary disease (COPD) affects 10 to 14 millionindividuals in the United States and is also characterized by mucusaccumulation in airway lumens and metaplasia of mucus secreting gobletcells. See, e.g. Celli et al., Am J Respir Crit Care Med 152, S177-S210(1995). Hence, there is a need to develop new ways to treat cysticfibrosis and chronic obstructive pulmonary disease.

[0004] In cystic fibrosis several functions of airway epithelia areabnormal, and deficiencies in both Cl³¹ transport and Na³⁰ absorptionare well documented. See, e.g. Knowles et al., Science 221, 1067 (1983);Knowles et al., J. Clin. Invest. 71, 1410 (1983). It would be extremelyuseful to provide a mouse model of cystic fibrosis and chronicobstructive pulmonary disease so that treatment options to improve mucusclearance in vivo could be more vigorously pursued. Unfortunately, priorefforts to develop a mouse model of cystic fibrosis produced animalsthat did not develop spontaneous lung disease. See, e.g., B. Grubb andR. Boucher, Physiological Reviews 79, S193-S214 (1999). Accordingly,there is a need for new approaches to solving the problem of providingan animal model for cystic fibrosis or chronic obstructive pulmonarydisease.

SUMMARY OF THE INVENTION

[0005] A first aspect of the present invention is a recombinant nucleicacid comprising a promoter operably linked to a heterologous nucleicacid (such as a DNA or RNA) encoding an epithelial sodium channel αsubunit, β subunit, and/or γ subunit, wherein the promoter constructdirects expression of the epithelial sodium channel α subunit, βsubunit, and/or γ subunit in lung epithelial cells. In one preferredembodiment, the heterologous nucleic acid encodes an ENaC β subunit anddirects expression of that subunit in lung epithelial cells.

[0006] A second aspect of the present invention is a host cell(particularly a mammalian host cell) containing a recombinant nucleicacid as described above.

[0007] A third aspect of the present invention is a method of making anon-human transgenic animal, comprising introducing a nucleic acid asdescribed above into an egg cell or embryonic cell of a non-humanembryo, implanting said egg or embryonic cell into a compatible femalehost, and raising said egg or embryonic cell to viability in said femalehost.

[0008] A further aspect of the present invention is a nonhumantransgenic mammal (e.g., mouse, rat, pig, monkey) whose genome comprisesa promoter construct operably linked to a heterologous DNA encoding anepithelial sodium channel β subunit, wherein the promoter constructdirects expression of the epithelial sodium channel β subunit in lungepithelial cells of the mammal, and wherein the transgenic mammal hasincreased lung mucus retention as compared to the correspondingwild-type mammal.

[0009] In one embodiment of the foregoing, the genome of the mammalpreferably comprises a promoter construct operably linked to aheterologous DNA encoding an epithelial sodium channel β subunit,wherein the promoter construct directs expression of the epithelialsodium channel β subunit in lung epithelial cells of the

[0010] In one embodiment of the foregoing, the genome of the mammalfurther comprises a promoter construct operably linked to a heterologousDNA encoding an epithelial sodium channel α subunit, wherein thepromoter construct directs expression of the epithelial sodium channel αsubunit in lung epithelial cells of the mammal.

[0011] In another embodiment of the foregoing, the genome of the mammalfurther comprises a promoter construct operably linked to a heterologousDNA encoding an epithelial sodium channel γ subunit, wherein thepromoter construct directs expression of the epithelial sodium channel γsubunit in lung epithelial cells of the mammal.

[0012] In some preferred embodiments, the nonhuman transgenic mammal hasincreased lung mucus plugging as compared to the corresponding wild-type(e.g., non-transformed) mammal, and/or increased mortality at 30 days ofage as compared to the corresponding wild-type mammal, and/or exhibits acystic fibrosis or chronic obstructive pulmonary disease phenotype notexhibited by the corresponding wild-type mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1: Transgenic constructs for airway specific overexpressionof α, β and γ mENaC.

[0014]FIG. 2. Detection of α, β and γ mENaC transgenes by Southernblotting.

[0015]FIG. 3. Detection of α, β and γ mENaC transgenes by PCR fromgenomic tail DNA.

[0016]FIG. 4. Detection of expression of α, β and γ mENaC tg transcriptsin mouse airways by RT-PCR from tracheal tissue.

[0017]FIG. 5. Quantitation of expression levels of α(A), β(B), and γmENaC (C) transgenes relative to WT in tracheal tissues of transgenepositive mouse lines relative to wild type littermate controls, asdetermined by quantitative RT-PCR.

[0018]FIG. 6. Survival curves of α-, β- and γ-mENaC transgene positive(tg/−) mice and wild-type (−/−) littermate controls.

[0019]FIG. 7. Lung histology of a β mENaC transgene positive mouse thatdied spontaneously on day 20. H&E staining shows occlusion of airwaysthroughout the lung (a,c). Scale bars=200 mm.

[0020]FIG. 8. Lung histology of β mENaC transgene positive mice thatwere euthanized at 3 (a,b) and 28 (c,d) days of age. All scale bars=200mm.

[0021]FIG. 9. Lung histology of a βtg mouse that was euthanized on day28. Scale bar=200 mm.

[0022]FIG. 10. Basal bioelectric properties of tracheal tissues of (A)neonatal (3-4 day old) and (B) adult (6 week old) αtg, βtg, and γtg ENaCover-expressing mice. *P<0.05 compared to WT. Data are means±SEM, n=3.

[0023]FIG. 11. Summary of amiloride-sensitive I_(sc) in tracheal tissuesof (A) neonate (3-4 days old) and (B) adult (6 week old) αtg, βtg, andγtg over-expressing mice. *P<0.05 compared to WT. Data are means±SEM,n=3.

[0024]FIG. 12. Summary of (A) forskolin-induced I_(sc) and (B)UTP-induced I_(sc) in tracheal tissues of adult (6 week old) αtg, βtg,and γtg over-expressing mice. Data are means±SEM, n=3.

[0025]FIG. 13. Comparison of PCL height in OsO4/PFC fixed airwaysbetween β-mENaC over-expressing mice and WT littermate controls by TEM.(A) In WT animals the PCL is covered by a thin (0.3-0.4 mm) mucus film(electron dense epiphase). (B) Summary of PCL height in the trachea andbronchi of wild-type and b_(tg) over-expressing mice.

[0026]FIG. 14. Airway mucus cast removed from the trachea of a 5 day oldβ transgenic mouse pup. Scale bar=0.1 mm.

[0027]FIG. 15. Mucociliary clearance (MCC) in the lower airways of WTand β tg mice determined using in vivo microdialysis. Data shown aremeans±SEM, with the number of mice studied shown in parentheses. *p<0.03 compared to male WT, ** p<0.01 compared to female WT.

[0028]FIG. 16. Histology (H&E and AB-PAS staining) on lungs from WT(panels A,C) and β tg (B,D) mice 72 hrs after nasal instillation ofPseudomonas (strain PAO1). All scale bars=100 um.

[0029]FIG. 17. Summary of quantitative bacteriology on lung homogenatesfrom WT (open circles) and β tg (closed circles) mice after 72 hrsfollowing challenge with Pseudomonas aeruginosa by trachealinstillation. * p<0.01 compared to WT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] “Mammal” as used herein refers to non-human mammals such as mice,sheep, pigs, rats, and cows.

[0031] “Operatively associated” or “operatively linked” as used hereinwith respect to nucleic acids indicates that the two segments of anucleic acid functionally interact with one another in their intendedmanner in a host cell. For example, a promoter is operatively associatedwith a nucleic acid of interest when it facilitates or permits thetranscription of the nucleic acid in a host cell; a locus control regionis operatively associated with a promoter when it enhances the activityof the promoter to provide high level nucleic acid transcription inspecific tissues (i.e., tissue-specific expression of the associatednucleic acid).

[0032] “Epithelium sodium channel” or “ENaC” as used herein, may be ofany source, including but not limited to human, mouse, rat, or othermammalian source. The epithelium sodium channel α, β, and γ subunits andnucleic acids encoding the same are known. The mouse alpha ENaC subunitis described at GENBANK Accession No. AF112185; the mouse beta ENaCsubunit is described at GENBANK Accession No. NM_(—)011325; the mousegamma ENaC subunit is described at GENBANK Accession No. NM_(—)011326;the rat alpha ENaC subunit is described at GENBANK Accession No. X70497;the rat beta ENaC subunit is described at GENBANK Accession No. X77932;the rat gamma ENaC subunit is described at GENBANK Accession No. X77933;the human alpha ENaC subunit is described at GENBANK Accession No.NM_(—)001038; the human beta ENaC subunit is described at GENBANKAccession No. NM_(—)000336; and the human gamma ENaC subunit isdescribed at GENBANK Accession No. NM_(—)001039, the disclosures of allof which are incorporated by reference herein in their entirety. Inaddition, all 3 ENaC subunits in human and rat are described in C.Canessa et al., Epithelial sodium channel related to proteins involvedin neurodegeneration, Nature 361 (6411), 467-470 (1993); C. Canessa etal., Amiloride-sensitive epithelial Na+ channel is made of threehomologous subunits, Nature 367 (6462), 463-467 (1994); N. Voilley etal., The lung amiloride-sensitive Na+ channel: biophysical properties,pharmacology, ontogenesis, and molecular cloning, Proc. Natl. Acad. Sci.U.S.A. 91 (1), 247-251 (1994); N. Voilley et al., Cloning, chromosomallocalization, and physical linkage of the beta and gamma subunits(SCNN1B and SCNN1G) of the human epithelial amiloride-sensitive sodiumchannel, Genomics 28 (3), 560-565 (1995), the disclosures of all ofwhich are incorporated herein by reference in their entirety.

[0033] “Promoter” as used herein refers to any type of promoter,including constitutive promoters or regulated promoters, unlessotherwise specified. Preferably the promoter is one which selectively orpreferentially directs expression of the epithelial sodium channel βsubunit in lung epithelial cells (e.g., a promoter that is airway cellspecific, ciliated cells specific or Clara cell specific). Examples ofsuch promoters include, but are not limited to, the Clara cell secretoryprotein (CCSP) promoter, the surfactant protein C promoter (S. Glasseret al., Genetic element from human surfactant protein SP-C gene confersbronchiolar-alveolar cell specificity in transgenic mice, Am. J.Physiol. 261, L349-L356 (1991)), the cytokeratin 18 promoter (GENBANKAccession No. AF179904 M24842 M19353 X12799; Y. Chow et al., Developmentof an epithelium-specific expression cassette with human DNA regulatoryelements for transgene expression in lung airways, Proc Natl Acad Sci US A 1997 Dec 23;94(26):14695-700), and the human forkhead homologue 4promoter (S. Brody et al., Structural characterization of the mouse Hfh4gene, a developmentally regulated forkhead family member, Genomics45(3):509-18 (1997)).

[0034] Such promoters may be of any source, including but not limited tohuman, mouse, rat, rabbit, primate or other mammalian source. The ratCCSP promoter is currently preferred and is described in GENBANKAccession No. X51318 (G. Hagen et al., Tissue-specific expression,hormonal regulation and 5′-flanking gene region of the rat Clara cell 10kDa protein: comparison to rabbit uteroglobin, Nucleic Acids Res. 18(10), 2939-2946 (1990); B. Hackett and J. Gitlin, 5′ flanking region ofthe Clara cell secretory protein gene specifies a unique temporal andspatial pattern of gene expression in the developing pulmonaryepithelium, Am J Respir Cell Mol Biol. 11(2):123-9 (1994).

[0035] “Host cell” as used herein refers to any type of cell into whicha recombinant or heterologous nucleic acid as described herein has beeninserted. Such cells are generally eukaryotic cells, particularlymammalian cells, including pig, cow, sheep, and mouse cells.

[0036] “Increased lung mucus retention” as used herein is defined orcharacterized by the spontaneous presence of airway mucus in amountsthat are readily detected by light microscopy after staining for acidand neutral mucins (e.g. with Alcian Blue and Periodic Acid Schiffstaining) in transgenic animals. In contrast, wild-type animals produceonly very scant spontaneous airway mucus that is not substantiallydetected by light microscopy.

[0037] “Increased mucus plugging” as used herein is defined orcharacterized by spontaneous formation of complete (plugs) or partial(plaques) obstruction of airway lumens with mucus in transgenic animals.Increased inflammation may accompany increased mucus plugging.Spontaneous mucus plug formation does not appreciably occur in wild-typeanimals.

[0038] “Increased mortality ” as used herein is defined or characterizedby an increase in death rates of transgenic animals between birth andadulthood compared to wild-type littermates. Spontaneous mortality inwild type mice in the first 8 weeks of life does not exceed ˜10%. Incontrast, mortality rates of different β-ENaC overexpressing micerepresenting certain embodiments of the present invention were ≧30%.

[0039] “Cystic fibrosis and chronic obstructive pulmonary diseasephenotype” as used herein is defined as spontaneous mucus accumulationand goblet cell metaplasia, leading to the formation of mucus plugs andplaques, which in turn results in airway obstruction (e.g., as found orseen in chronic bronchitis and asthma).

[0040] The disclosures of all United States patent references citedherein are to be incorporated by reference herein in their entirety.

1. Nucleic Acid Constructs and Transformed Host Cells

[0041] The production of recombinant nucleic acids, vectors, transformedhost cells, proteins and protein fragments by genetic engineering iswell known. See, e.g., U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6line 3 to Col. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. atCol. 4 line 38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling atCol. 3 line 26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 toWallner at Col. 6 line 8 to Col. 8 line 59.

[0042] As noted above, a further aspect of the present invention is anisolated nucleic acid construct comprising at least one locus controlregion as described above operatively associated with a promoter. Thepromoter may be a heterologous promoter or homologous promoter, andwhere a homologous promoter the isolated nucleic acid may or may notinclude intervening segments.

[0043] A vector is a replicable nucleic acid construct or a nucleic acidconstruct used to insert particular nucleic acid constructs into a hostcell. Vectors are used herein either to amplify nucleic acid constructsof the present invention or insert the constructs into a host cell oranimal. Vectors comprise plasmids, viruses (e.g., adenovirus,cytomegalovirus, retroviruses), phage, and linear nucleic acids such asintegratable DNA fragments (i.e., fragments integratable into the hostgenome by recombination). The vector may replicate and functionindependently of the host genome or may in some instances, integrateinto the genome itself.

[0044] If desired, the vector may optionally contain flanking nucleicsequences that direct site-specific homologous recombination. The use offlanking DNA sequence to permit homologous recombination into a desiredgenetic locus is known in the art. At present it is preferred that up toseveral kilobases or more of flanking DNA corresponding to thechromosomal insertion site be present in the vector on both sides of theencoding sequence (or any other sequence of this invention to beinserted into a chromosomal location by homologous recombination) toassure precise replacement of chromosomal sequences with the exogenousDNA. See e.g. Deng et al, 1993, Mol. Cell. Biol 13(4):2134-40; Deng etal, 1992, Mol Cell Biol 12(8):3365-71; and Thomas et al, 1992, Mol CellBiol 12(7):2919-23. It should also be noted that the cell of thisinvention may contain multiple copies of the gene of interest.

[0045] Transformed host cells are cells which have been transformed ortransfected with vectors containing nucleic acid constructs of theinvention and may or may not transcribe or translate the operativelyassociated nucleic acid of interest.

2. Transgenic Animals and Methods of Making

[0046] The production of transgenic animals is well known and can becarried out in accordance with known techniques or variations thereofwhich will be apparent to those skilled in the art, for example asdisclosed in: U.S. Pat. No. 6,344,596 to W. Velander et al. (AmericanRed Cross); U.S. Pat. No. 6,339,183 to T. T. Sun (New York University);U.S. Pat. No. 6,331,658 to D. Cooper and E. Koren; U.S. Pat. No.6,255,554 to H. Lubon et al. (American National Red Cross; VirginiaPolytechnic Institute); U.S. Pat. No. 6,204,431 to P. Prieto et al.(Abbott Laboratories); U.S. Pat. No. 6,166,288 to L. Diamond et al.(Nextran Inc., Princeton, N.J.); U.S. Pat. No. 5,959,171 to J. M.Hyttinin et al. (Pharming BV); U.S. Pat. No. 5,880,327 to H. Lubon etal. (American Red Cross); U.S. Pat. No. 5,639,457 to G. Brem; U.S. Pat.No. 5,639,940 to I. Garner et al. (Pharmaceutical Proteins Ltd.;Zymogenetics Inc); U.S. Pat. No. 5,589,604 to W. Drohan et al. (AmericanRed Cross); U.S. Pat. No. 5,602,306 to Townes et al. (UAB ResearchFoundation); U.S. Pat. No. 4,736,866 to Leder and Stewart (Harvard); andU.S. Pat. No. 4,873,316 to Meade and Lonberg (Biogen).

[0047] For example, animals may be produced as described in U.S. Pat.No. 5,859,310 to Bujard et al., at column 17, which generally describesmethods in which a transgenic animal which contains in its genome thenucleic acid of interest is produced by the following steps: (1) Achimeric DNA sequence is prepared where a Tc responsive promoterelement, (comprising at least one tet operator and a minimal promoter)is cloned 5′ of the DNA sequences encoding the endogenous gene ofinterest. (2) The chimeric DNA sequence (called also “the chimerictransgene”) is then injected into a fertilized egg, which is implantedinto a pseudopregnant recipient mother and allowed to develop into anadult animal. In particular, a few hundred DNA molecules are injectedinto the pro-nucleus of a fertilized one cell egg. The microinjectedeggs are then transferred into the oviducts of pseudopregnant fostermothers and allowed to develop. See generally Brinster et al. Proc.Natl. Acad. Sci. U.S.A. Vol. 83:9065-9069 (1986). Breeding of animalsresulting from this process produces offspring containing the chimerictransgene. As will be appreciated, the particular breeding strategydepends on factors such as the nucleic acid of interest and the animalinto which it is inserted. Animals of the present invention can beproduced by substantially the same techniques, by injecting nucleic acidconstructs of the present invention.

[0048] U.S. Pat. No. 4,873,191 to Wagner and Hoppe (Ohio University)describes a method of obtaining a mammal characterized as having aplurality of cells containing exogenous genetic material, the materialincluding at least one gene and a control sequence operably associatedtherewith, which, under predetermined conditions, expresses the geneunder the control of the control sequence in a cell of the mammal. Themethod, which may also be used to make animals of the present invention,comprises: (a) introducing exogenous genetic material into a pronucleusof a mammalian zygote by microinjection, the zygote being capable ofdevelopment into a mammal, the genetic material including at least onegene and a control sequence operably associated therewith, therebyobtaining a genetically transformed zygote; (b) transplanting an embryoderived from the genetically transformed zygote into a pseudopregnantfemale capable of bearing the embryo to term; and (c) allowing theembryo to develop to term; where the gene and control sequence areselected so that the gene is not activated in such manner and degree aswould prevent normal development of the embryo to term. Again, animalsof the present invention can be produced by substantially the sametechniques, by introducing nucleic acid constructs of the presentinvention into the pronucleus zygote by microinjection.

[0049] U.S. Pat. No. 6,369,294 to J. Piedrahata and F. Bazer (Texas A&MUniversity System) describes a method of producing a transgenic pig thatmay be used to carry out the present invention. The method comprises (a)introducing a selected DNA segment into a cell culture comprisingporcine primordial germ cells to obtain candidate porcine primordialgerm cells that contain the selected DNA segment; (b) plating thecandidate porcine primordial germ cells that contain the selected DNAsegment on feeder cells (the feeder cells preferably at a density ofbetween about 2.5×10⁵ cells/cm² and about 10⁶ cells/m²), in a culturemedium comprising an effective amount of basic fibroblast growth factorand an apoptosis inhibitor, to obtain undifferentiated porcineprimordial germ cells that contain the selected DNA segment; and (c)generating a transgenic pig from the undifferentiated porcine primordialgerm cells that contain the selected DNA segment, wherein the selectedDNA segment is contained and expressed in somatic and germ cells of thetransgenic pig. Animals of the present invention can be produced in likemanner by utilizing the nucleic acid constructs described herein as theselected DNA segment.

[0050] U.S. Pat. No. 5,573,933 to R. Seamark and J. Wells (Luminis Pty.,Ltd.) describes a method for preparing a transgenic pig that may be usedto carry out the present invention. The method comprises introducing aplasmid expression vector or a linerized insert therefrom comprising thefirst, second and third DNA sequences into the male pronucleus of thefertilized pig ovum prior to fusion with the female nucleus to form asingle cell embryo; and, subsequently implanting the ovum into a femalepig and allowing the embryo, resulting from introduction of the plasmidcloning vector into the ovum, to develop to maturity. Animals of thepresent invention can be produced in like manner as described therein.

3. Applications of the Invention

[0051] The present invention provides a method of screening compoundsfor activity in treating lung disease. The method comprises the stepsof: providing a nonhuman transgenic mammal as described herein;administering a test compound to the subject, and determining the effectof the test compound on susceptibility to airway disease in the animal,a decrease in susceptibility to airway disease in the animal indicatingthat the test compound may be useful in treating lung disease. The testcompound may be administered by any suitable technique, including butnot limited to aerosol administration, parenteral administration(subcutaneous injection, intramuscular injection, intraveneousinjection, etc.), transdermal administration, etc. Decrease insusceptibility to airway disease in the animal may be determineddirectly or indirectly by any suitable technique, including but notlimited to examining any of the phenotypes exhibited by the transgenicanimal as described above and examining the animal for an improvement insuch phenotypes (e.g., a partial or complete return to the phenotypeexhibited by the corresponding wild-type animal).

[0052] Such screens may be utilized for screening for compoundsbeneficial in treating any type of lung disease, particularly chronicobstructive pulmonary disease or cystic fibrosis. Any category ofcompound may be screened, examples including but not limited toantibiotics, osmolites, sodium channel blockers, P2Y₂ receptor agonists,ENaC regulators including CAP-1 protease, anti-inflammatory compoundsincluding non-steroidal anti-inflammatory compounds, agents blockinggoblet cell metaplasia and mucus hypersecretion such as epithelialgrowth factor (EGFR) blockers, IL-13 and IL-13 receptor blockers,blockers of the STAT6 signalling pathway, and Lomucin (produced byGenera, Research Triangle Park North Carolina, USA), anti-mucussecretion compounds such as MARCKS protein (Biomarcks, RTP),P2Y₂-receptor antagonists, mucolytic agents such as N-acetyl-cystein andderivatives thereof, etc.

[0053] Animals of the present invention are also useful for theidentification of genes associated with airway inflammation and gobletcell metaplasia.

[0054] The present invention is explained in greater detail in thefollowing non-limiting Examples.

EXAMPLE 1 Preparation of Transgenic Mice

[0055] Transgenic constructs for airway specific overexpression of α, βand γ mENaC. are schematically illustrated in FIG. 1. Transgenic mouselines overexpressing α-, β-, or γ-ENaC under the control of the CCSPpromoter were produced as follows: To generate plasmids for individualENaC subunits we made use of pTG1 plasmid (obtained from Dr. RandyThresher, Animal Model Core facility, UNC) containing two multiplecloning sites (MCS1 and MCS2), separated by an intron, and followed by aSV40 polyadenylation signal.

[0056] The rat CCSP promoter (rCCSP) was obtained from Dr. J D Gitlin JD (for reference see: Hackett B P and Gitlin J D. Cell-specificexpression of a Clara cell secretory, protein-human at growth hormonegene in the bronchiolar epithelium of transgenic mice. Proc Natl AcadSci U S A 1992 Oct 1;89(19):9079-83), amplified from plasmid by PCR, andcloned into MCS1 of pTG1.

[0057] ENaC subunits from mouse (α-, β-, γ-mENaC) were obtained from Dr.Tomas Kleyman (for reference see: Ahn Y J, Brooker D R, Kosari F, HarteB J, Li J, Mackler S A, Kleyman T R. Cloning and functional expressionof the mouse epithelial sodium channel. Am J Physiol 1999 Jul; 277(1 Pt2):F121-9), amplified from plasmid by PCR, and the coding sequences ofindividual subunits (α, β, or γ-ENaC) were cloned into MCS2 ofpTG1/CCSP, respectively.

[0058] The α-construct contained nucleotides 6 to 2131 of GENBANKAccession No. AF1112185 (mouse alpha ENaC subunit); the β-constructcontained nucleotides 30 to 1952 of GENBANK Accession No. NM_(—)011325(mouse beta ENaC subunit); and the γ-construct contained nucleotides 70to 2037 of GENBANK Accession No. NM_(—)011326 (mouse gamma ENaCsubunit). Note that constructs containing more of the untranslatedsequences are expected to work equally well, as long as they contain thecomplete or sufficient coding sequence.

[0059] Three unique SV40 sequences of ˜280 bp in length were generatedby PCR from pUCSV40-B2E plasmid (purchased from American Type CultureCollection, ATCC, manassas, Va.), and inserted into transgenicconstructs at the 3′ end of individual ENaC subunits, respectively, forpurposes of detection of transgene mRNA expression (SV40tag I-III).Sequences of rCCSP, α-, β-, γ-mENaC and SV40tag I-III were verified byautomatic sequencing. Individual transgenic constructs were isolated byrestriction enzyme digestion, purified and injected into fertilized eggsby means of pronuclear injection (performed by Dr. Randy Thresher,Animal Model Core facility, UNC). After DNA injection, fertilized eggswere implanted into pseudopregnant recipient mothers. Animals generatedby this process were genotyped to identify transgene positive foundersand individual lines of transgenics were bred from these founders.

EXAMPLE 2 Detection of Transgenes in Transgenic Mice

[0060] The detection of α, β and γ mENaC transgenes by Southern blottingis shown in FIG. 2. Transgenic (Tg) positive mice were identified byhybridization with P³²-labeled transgene-specific probes, resulting in a2.7 kb band for αtg, a 0.6 kb band for βtg and a 2.6 kb band for γtg.

[0061] The detection of α, β and γ mENaC transgenes by PCR from genomictail DNA is shown in FIG. 3. Length of different PCR products: αtg 228bp vs αwt˜650 bp; βtg 255 bp vs βwt˜350 bp; γtg 334 bp vs γwt 1200 bp.

[0062] The detection of expression of α, β and γ mENaC tg transcripts inmouse airways by RT-PCR from tracheal tissue is shown in FIG. 4.Expression of tg transcripts in tissue of tg positive mice was detectedby amplification of PCR products of the expected size after reversetranscription of total RNA and using tg specific primer pairs.

[0063] A quantitation of expression levels of α(A), β(B), and γ mENaC(C) transgenes relative to WT in tracheal tissues of transgene positivemouse lines relative to wild type littermate controls, as determined byquantitative RT-PCR is shown in FIG. 5.

EXAMPLE 3 Survival of Transgenic Mice

[0064] Survival curves of α-, β- and γ-mENaC transgene positive (tg/−)mice and wild-type (−/−) littermate controls are shown in FIG. 6. (A) α,(B) β, and (C) γENaC tg mice. All tg mice were heterozygous for thetransgene.

EXAMPLE 4 Lung Histology from Transgenic Mice

[0065] Lung histology of a 13 mENaC transgene positive mouse that diedspontaneously on day 20 is shown in FIG. 7. H&E staining shows occlusionof airways throughout the lung (a,c). AB-PAS staining identifies theintraluminal material as mucus. (b,d). Scale bars=200 mm.

[0066] Lung histology of βmENaC transgene positive mice that wereeuthanized at 3 (a,b) and 28 (c,d) days of age is shown in FIG. 8. Lunghistology in βtg neonates is normal (ai,bi) with no evidence of mucusretention or goblet cell metaplasia (aii,,bii). Older mice exhibit mucusretention with various degrees of airway obstruction, from narrowing ofairways to complete plugging, and goblet cell metaplasia (GCM)(cii,dii). H&E stain in (ai,bi,ci,di) and AB-PAS in (aii,bii,cii,dii).All scale bars=200 mm.

[0067] Lung histology of a βtg mouse that was euthanized on day 28 isshown in FIG. 9. AB-PAS staining shows GCM along different regions ofthe bronchial tree, ranging from large airways to terminal bronchioles(AB-PAS). Scale bar=200 mm.

EXAMPLE 5 Functional Properties of Tracheal Tissue from Transgenic Mice

[0068] Basal bioelectric properties of tracheal tissues of (A) neonatal(3-4 day old) and (B) adult (6 week old) αtg, βtg, and γtg ENaCover-expressing mice are shown in FIG. 10. Electrogenic ion transport issignificantly increased in βtg, but not in αtg or γtg mice. Twodifferent transgenic founder lines were tested per transgene. *P<0.05compared to WT. Data are means±SEM, n=3.

[0069] A summary of amiloride-sensitive I_(sc) in tracheal tissues of(A) neonate (3-4 days old) and (B) adult (6 week old) αtg, βtg, and γtgover-expressing mice is shown in FIG. 11. Two different transgenicfounder lines were tested per transgene. Electrogenic Na⁺ transport issignificantly increased in βtg, but not in αtg or γtg mice. *P<0.05compared to WT. Data are means±SEM, n=3.

[0070] A summary of (A) forskolin-induced I_(sc) and (B) UTP-inducedI_(sc) in tracheal tissues of adult (6 week old) αtg, βtg, and γtgover-expressing mice is given in FIG. 12. Experiments were performed inthe presence of amiloride. Two different transgenic founder lines weretested per transgene. Forskolin- and/or UTP-induced Cl− secretion wasnot different in any of the transgenic lines. Data are means±SEM, n=3.

[0071] A comparison of PCL height in OsO4/PFC fixed airways betweenβ-mENaC over-expressing mice and WT littermate controls by TEM is givenin FIG. 13. (A) In WT animals the PCL is covered by a thin (0.3-0.4 mm)mucus film (electron dense epiphase). Airways of βtg over-expressingmice show depletion of PCL, reduced PCL height and bending of cilia.Furthermore mucus accumulation is found in some airway regions of βtgmice, but not in WT. (B) Summary of PCL height in the trachea andbronchi of wild-type and btg over-expressing mice.

EXAMPLE 6 Mucus Clearance from Transgenic Mice

[0072] An airway mucus cast removed from the trachea of a 5 day old btransgenic mouse pup is shown in FIG. 14. An air bubble, the dark image,is clearly visible trapped within and distending the lumen of the cast.Scale bar=0.1 mm.

[0073] Mucociliary clearance (MCC) in the lower airways of WT and βtgmice as determined using in vivo microdialysis is shown in FIG. 15. Datashown are means±SEM, with the number of mice studied shown inparentheses. MCC is significantly reduced in β tg mice. * p<0.03compared to male WT, ** p<0.01 compared to female WT.

EXAMPLE 7 Response of Transgenic Mice to Lung Bacterial Challenge

[0074] Histology (H&E and AB-PAS staining) on lungs from WT (panels A,C)and βtg (B,D) mice 72 hrs after nasal instillation of Pseudomonas(strain PAO1) is shown in FIG. 16. Intraluminal airway infection wasdetected in βtg mice (B,D), but not in WT mice (A,C). Airway infectionin βtg mice was characterized by infiltration of mucus plugs/plaqueswith neutrophils and macrophages (B.D). No such lesions were detected inany of the WT mice (A,C). H&E stain (A,B) and AB-PAS (C,D). All scalebars=100 um.

[0075] A summary of quantitative bacteriology on lung homogenates fromWT (open circles) and βtg (closed circles) mice after 72 hrs followingchallenge with Pseudomonas aeruginosa by tracheal instillation is givenin FIG. 17. Bacterial clearance was significantly reduced in b tgmice. * p<0.01 compared to WT.

[0076] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A recombinant nucleic acid comprising apromoter operably linked to a heterologous nucleic acid encoding anepithelial sodium channel β subunit, wherein said promoter constructdirects expression of the epithelial sodium channel β subunit in lungepithelial cells.
 2. The recombinant nucleic acid according to claim 1,wherein said promoter is selected from the group consisting of the CCSPpromoter, the surfactant protein C promoter, the cytokeratin 18promoter, and the human forkhead homologue 4 promoter.
 3. Therecombinant nucleic acid according to claim 1, wherein said promoter isthe CCSP promoter.
 4. A host cell containing a recombinant nucleic acidaccording to claim
 1. 5. A nonhuman transgenic mammal whose genomecomprises a promoter construct operably linked to a heterologous nucleicacid encoding an epithelial sodium channel β subunit, wherein saidpromoter construct directs expression of the epithelial sodium channel βsubunit in lung epithelial cells of said animal, and wherein saidtransgenic mammal has increased lung mucus retention as compared to thecorresponding wild-type mammal.
 6. The nonhuman transgenic mammal ofclaim 5, wherein said promoter is selected from the group consisting ofthe CCSP promoter, the surfactant protein C promoter, the cytokeratin 18promoter, and the human forkhead homologue 4 promoter.
 7. The nonhumantransgenic mammal of claim 5, wherein the genome of said mammal furthercomprises a promoter construct operably linked to a heterologous nucleicacid encoding an epithelial sodium channel α subunit, wherein saidpromoter construct directs expression of the epithelial sodium channel αsubunit in lung epithelial cells of said animal.
 8. The nonhumantransgenic mammal of claim 5, wherein the genome of said mammal furthercomprises a promoter construct operably linked to a heterologous nucleicacid encoding an epithelial sodium channel γ subunit, wherein saidpromoter construct directs expression of the epithelial sodium channel γsubunit in lung epithelial cells of said animal.
 9. The nonhumantransgenic mammal of claim 5, wherein said mammal is selected from thegroup consisting of mice, rats, pigs, and monkeys.
 10. The nonhumantransgenic mammal of claim 5, wherein said mammal is a mouse.
 11. Thenonhuman transgenic mammal of claim 5, wherein said mammal has increasedlung mucus plugging and airway inflammation as compared to thecorresponding wild-type mammal.
 12. The nonhuman transgenic mammal ofclaim 5, wherein said transgenic mammal has increased mortality at 30days of age as compared to the corresponding wild-type mammal.
 13. Thenonhuman transgenic mammal of claim 5, wherein said transgenic mammalexhibits a cystic fibrosis or chronic obstructive pulmonary diseasephenotype not exhibited by the corresponding wild-type mammal.
 14. Amethod of screening compounds for activity in treating lung disease,comprising: providing a nonhuman transgenic mammal whose genomecomprises a promoter construct operably linked to a heterologous DNAencoding an epithelial sodium channel β subunit, wherein said promoterconstruct directs expression of the epithelial sodium channel β subunitin lung epithelial cells of said animal, and wherein said transgenicmammal has increased lung mucus retention as compared to thecorresponding wild-type mammal; and then administering a test compoundto said subject, and determining the effect of said test compound onsusceptibility to airway disease in said animal, a decrease insusceptibility to airway disease in said animal indicating that saidtest compound may be useful in treating lung disease.
 15. The method ofclaim 14, wherein said lung disease is chronic obstructive pulmonarydisease or cystic fibrosis.
 16. The method of claim 14, wherein saidlung disease is chronic bronchitis.
 17. The method of claim 14, whereinsaid lung disease is asthma.
 18. The method of claim 14, wherein saidtest compound is an antibiotic.
 19. The method of claim 14, wherein saidtest compound is an osmolite.
 20. The method of claim 14, wherein saidtest compound is a sodium channel blocker.
 21. The method of claim 14,wherein said test Compound is a P2Y₂ receptor agonist.
 22. The method ofclaim 14, wherein said test compound is an ENaC regulator.
 23. Themethod of claim 14, wherein said test Compound is an anti-inflammatoryagent.
 24. The method of claim 14, wherein said test compound is amucolytic agent.